Structure of the [Ca]E2P intermediate of Ca²⁺-ATPase 1 from Listeria monocytogenes

TY - JOUR

T1 - Structure of the [Ca]E2P intermediate of Ca2+-ATPase 1 from Listeria monocytogenes

AU - Basse Hansen, Sara

AU - Flygaard, Rasmus Kock

AU - Kjaergaard, Magnus

AU - Nissen, Poul

PY - 2025/4/7

Y1 - 2025/4/7

N2 - Active transport by P-type Ca2+-ATPases maintain internal calcium stores and a low cytosolic calcium concentration. Structural studies of mammalian sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) have revealed several steps of the transport cycle, but a calcium-releasing intermediate has remained elusive. Single-molecule FRET studies of the bacterial Ca2+-ATPase LMCA1 revealed an intermediate of the transition between so-called [Ca]E1P and E2P states and suggested that calcium release from this intermediate was the essentially irreversible step of transport. Here, we present a 3.5 Å resolution cryo-EM structure for a four-glycine insertion mutant of LMCA1 in a lipid nanodisc obtained under conditions with calcium and ATP and adopting such an intermediate state, denoted [Ca]E2P. The cytosolic domains are positioned in the E2P-like conformation, while the calcium-binding transmembrane (TM) domain adopts a calcium-bound E1P-ADP-like conformation. Missing density for the E292 residue at the calcium site (the equivalent of SERCA1a E309) suggests flexibility and a site poised for calcium release and proton uptake. The structure suggests a mechanism where ADP release and re-organization of the cytoplasmic domains precede calcium release.

AB - Active transport by P-type Ca2+-ATPases maintain internal calcium stores and a low cytosolic calcium concentration. Structural studies of mammalian sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) have revealed several steps of the transport cycle, but a calcium-releasing intermediate has remained elusive. Single-molecule FRET studies of the bacterial Ca2+-ATPase LMCA1 revealed an intermediate of the transition between so-called [Ca]E1P and E2P states and suggested that calcium release from this intermediate was the essentially irreversible step of transport. Here, we present a 3.5 Å resolution cryo-EM structure for a four-glycine insertion mutant of LMCA1 in a lipid nanodisc obtained under conditions with calcium and ATP and adopting such an intermediate state, denoted [Ca]E2P. The cytosolic domains are positioned in the E2P-like conformation, while the calcium-binding transmembrane (TM) domain adopts a calcium-bound E1P-ADP-like conformation. Missing density for the E292 residue at the calcium site (the equivalent of SERCA1a E309) suggests flexibility and a site poised for calcium release and proton uptake. The structure suggests a mechanism where ADP release and re-organization of the cytoplasmic domains precede calcium release.

KW - Ca2+-ATPase

KW - Calcium Transport

KW - Calcium-bound Intermediate

KW - P-type ATPase

UR - http://www.scopus.com/inward/record.url?scp=86000081839&partnerID=8YFLogxK

U2 - 10.1038/s44319-025-00392-x

DO - 10.1038/s44319-025-00392-x

M3 - Journal article

C2 - 40016426

AN - SCOPUS:86000081839

SN - 1469-221X

VL - 26

SP - 1709

EP - 1723

JO - EMBO Reports

JF - EMBO Reports

IS - 7

M1 - eadd9742

ER -